Proviral location affects cognate peptide-induced virus production and immune recognition of 2 HIV-1-infected T cell clones
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE236837 | GEO | 2023/07/14
REPOSITORIES: GEO
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Project description:The reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure the reservoir of activatable proviruses should be removed, whereas permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. Latently HIV-1 infected cells were transduced with a zinc finger protein specifically binding to the HIV-1 promoter, producing a fluorescent signal as the viral transactivator Tat is recruited to the HIV-1 promoter. In these cells we assessed the proviral chromatin composition simultaneously with the proviral activation. By linking the Tat promoter recruitment to viral activation, we dissected the mechanisms of HIV-1 reactivation and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasts to the continuous production of viral proteins. As expected, promoter H3K4me3 led to massive expression of the provirus following T cell stimulation. However, the activation induced cell cycle arrest and death resulted in an expanded surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this model can be used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determine the effect of CBP/P300-inhibitor GNE049. We found that only active enhancers, associated with H3K4me1 and H3K27ac, efficiently recruit Tat, as GNE049 that specifically inhibits enhancer H3K27ac also depletes promoter Tat. Despite the absence of Tat, HIV-1 latency reversal still occurred. Single cell assessment of the chromatin composition of the latent HIV-1 proviruses revealed how T cell stimulation modulates the proviral activity and the subsequent fate of the infected cell.
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Project description:Our study reports the first genome-wide atlas of functional nodes that mediate proviral silencing in ESCs. It provides evidences for the comprehensive, interconnected and multi-layered genetic/epigenetic machineries by which ESCs maintain the repressive state of provirus and ERVs.
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Project description:Integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected cells. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. The “shock and kill” strategy aims to eradicate latent HIV by reactivating proviral gene expression followed by ART treatment. Gene specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising small guide RNAs (sgRNAs) with a nuclease deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the LTR promoter of HIV-1 and identified a “hotspot” for activation. We studied the functionality of activating sgRNAs to transcriptionally modulate the latent proviral genome across multiple different in vitro latency cell models including several J-Lat, ACH2 J1.1 and the CEM T cell model comprising a single clonal population of integrated mCherry-IRES-Tat from a full-length HIV LTR (LChIT). While we observed variable responses of latent cell models to well-characterized chemical stimuli, we detected consistent efficient activation of latent virus mediated by activator sgRNAs. In addition, transcriptome analysis of chemically treated cells revealed massive non-specific gene dysregulation whereas by comparison, dCas9-VP64/sgRNAs induced specific activation of the integrated provirus. In conclusion, we show the potential for CRISPR-mediated gene activation systems to provide enhanced efficiency and specificity in a targeted latency reactivation strategy. This represents a promising approach to a “functional cure” of HIV/AIDS.
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